﻿Podarkeopsischinensis sp. nov. (Annelida, Hesionidae) from southeastern China

﻿Abstract Podarkeopsischinensissp. nov. (Annelida, Hesionidae) is described based on specimens collected from the coast of southeast China. It is the first Podarkeopsis species described from the Indo-Pacific, although there are already nine valid Podarkeopsis species known from other parts of the world. This new species can be distinguished from the other Podarkeopsis species in having a palpostyle as long as the palpophore and double aciculae in both notopodia and neuropodia, and in bearing bifid furcate chaetae which have a smooth base on the shorter tine. A phylogenetic analysis based on the concatenated sequences of five gene fragments (COI, 16S rRNA, 18S rRNA, 28S rRNA, and histone H3) from 18 specimens of P.chinensissp. nov. showed that they formed a monophyletic clade that is sister to P.levifuscina. K2P genetic distances indicated that the four gene fragments (COI, 16S rRNA, 18S rRNA, and 28S rRNA) of P.chinensissp. nov. diverged from the corresponding sequences of the closest related species of Podarkeopsis in GenBank and BOLD Systems by 21.1–27.5%, 20.3–23.1%, 0.1–0.2%, and 2.1–3.2%. An identification key is provided for species in the genus Podarkeopsis.


Introduction
is a genus of small-bodied polychaetes in the family Hesionidae and currently comprises nine species (Read and Fauchald 2023), including three species from Europe, two species from the Pacific coast of the United States, one species from the Atlantic coast of the United States, one species from the Pacific coast of Central America, one species from the Atlantic coast of Central America, and one species from the Atlantic coast of South Africa (Fig. 1). Although Podarkeopsis species have unique morphological characteristics including having 10 triangular papillae, a pair of palps and three antennae placed towards the anterior prostomial margin, and eight pairs of tentacular cirri, its species diversity and phylogenetic relationships remain poorly understood, partially due to their minute size (a complete specimen is 5-18 mm long, 1-2 mm wide, and with 25-46 chaetigers), making them difficult to collect.
Currently, there are only a few studies with a description or specimen records of Podarkeopsis from Indo-Pacific. Imajima (2007) described P. brevipalpa (Hartmann-Schröder, 1959 and P. glabrus (Hartman, 1961) based on specimens collected from Japanese waters, which is the only morphological record of Podarkeopsis in the Indo-Pacific. However, this record is questionable, as the type locality of P. brevipalpa is the coast of El Salvador in Central America, and the type locality of P. glabrus is in southern California (Fig. 1). In addition, Podarkeopsis, mostly identified to the genus level, has been recorded in several ecological studies in the China seas (Shin 1998;Wang 2008;Li 2010;Du et al. 2011;Wang et al. 2017;Zhang 2017). Podarkeopsis galangani (a misspelling of P. galangaui Laubier, 1961) has been reported from Daya Bay, Guangdong, China, but this record is questionable, given that its type locality is the Mediterranean coast of France (Li 2010).
In this study, we describe and illustrate a new species, Podarkeopsis chinensis sp. nov., based on specimens collected from Daya Bay, Guangdong and ZhaoAn Bay, Fujian in southern China. We sequenced five gene fragments (COI,16S rRNA,18S rRNA,28S rRNA,and histone H3) to determine the phylogenetic position of the new species within Podarkeopsis.

Morphological analysis
Selected parapodia of the holotype (XMU-Pol-2021-105), paratype 10 (XMU-Pol-2021-209) and paratype 12 (XMU-Pol-2021-214) were dissected with iridectomy scissors and permanently mounted on slides for observation of their gross morphology and chaetae, as well as for photography. To observe the minute teeth inside the pharynx, the anterior region of the paratype 6 (XMU-Pol-2021-204), paratype 11 (XMU-Pol-2021-213), and paratype 13 (XMU-Pol-2021-215) were hyalinized with graded series of glycerol (30%, 60%, 100%), mounted on slides and compressed slowly with a cover glass. Photographs of the whole specimen and parapodia (with chaetae) were taken using a camera DMC5400 mounted on a Leica M165C stereomicroscope. Photographs were taken at different focuses and stacked into fully focused images using Helicon Focus v. 7 as described by Wang et al. (2018). The anterior region of the paratype 5 (XMU-Pol-2021-203) and paratype 12 (XMU-Pol-2021-214) were treated by critical point drying and fixed on a conductive adhesive for gold plating. Photographs of the anterior end were taken using Phenom ProX scanning electron microscope (SEM). The classification of the type of parapodia following Jarvis (2011): biramous with many emergent notochaetae; sub-biramous with few emergent notochaetae; sesquiramous with acicula inside cirrophore and without emergent chaetae.

DNA extraction, PCR amplification, and sequencing
Eighteen specimens of Podarkeopsis chinensis sp. nov. were used for DNA extraction. For each specimen, a few segments were dissected, and genomic DNA was extracted with a DNeasy Blood & Tissue Kit (QIAGEN). Five primer pairs were used to amplify corresponding gene fragments, viz., PolyLCO and Poly-HCO for the mitochondrial COI gene fragment (Carr et al. 2011), 16SAR-L and 16SBR-H for the mitochondrial 16S rRNA gene fragment (Palumbi et al. 1991), 1F and 9R for the nuclear 18S rRNA gene fragment (Glover et al. 2016); NLF184/21 and D3aR for the nuclear 28S rRNA gene fragment (Lenaers et al. 1989;Van der Auwera et al. 1994) and H3af and H3ar for the Histone H3 gene fragment (Colgan et al. 1998). The PCR protocol followed Zhang et al. (2018). The PCR products were checked by electrophoresis in a 2% agarose gel, and sequenced using Sanger sequencing at Xiamen Borui Biological Technology Co., Ltd.

Phylogenetic analyses
The sequences of the five gene fragments generated in this study, together with those of all corresponding Podarkeopsis and two Oxydromus species (outgroup) available in GenBank (https://www.ncbi.nlm.nih.gov/Genbank) and BOLD (http://www.barcodinglife.org), were used for phylogenetic analyses ( Table 1). The five gene sequences were aligned using the MUSCLE algorithm (Edgar 2004). The poorly aligned positions were removed with the Gblocks v. 0.91b plugged in PhyloSuite v. 1.2.3 (Zhang et al. 2020). Phylogenetic analyses were conducted using the maximum-likelihood (ML, IQ-TREE v.2.2.0 plugged in PhyloSuite) and Bayesian-inference (BI, MrBayes v.3.2.7a plugged in Phylo-Suite) methods. Specifically, the ML analysis with IQ-TREE (Nguyen et al. 2015) was conducted using the "ultrafast bootstrap" option with a bootstrap number of 10,000. The best-fit evolutionary model GTR+I+G was selected for the BI analysis using ModelFinder v. 2.2.0 based on the Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al. 2017). The BI analysis was conducted using MrBayes with Markov Chains run for 10,000,000 generations and topologies sampled every 1000 generations (Ronquist and Huelsenbeck 2003). The first 25% of trees were discarded as "burn-in" and the software Tracer v. 1.7.1 was used to check for the convergence of the trees (Rambaut et al. 2018). The resulting ML and BI trees were visualized using Figtree v. 1.4.4 (http://tree.bio. ed.ac.uk/software/figtree).
Habitat. Intertidal, shallow subtidal muddy sediment. Distribution. Podarkeopsis chinensis sp. nov. is currently known from Daya Bay, Guangdong and ZhaoAn Bay, Fujian, China. It is expected that this species is widely distributed along the coast of southeast China.

Phylogenetic analysis
The ML tree and BI tree, reconstructed based on the 3943-bp concatenated sequences, showed consistent topologies clustering the eight analyzed Podarkeopsis species within a single clade with high support values (BS = 100; BPP = 1) (Fig. 5). All 18 specimens of Podarkeopsis chinensis sp. nov. were clustered within a clade with high support values (BS= 100, BPP = 1), and P. chinensis sp. nov. was sister to P. levifuscina collected from Florida, USA

Discussion
Although Podarkeopsis is a common genus of polychaete in intertidal and subtidal sediments, it has attracted little attention, possibly due to its small size, fragilility, and usually incomplete condition of fixed specimens. This genus can be clearly distinguished from other hesionid genera by several remarkable char- acteristics, such as having the pharynx with 10 papillae on the anterior edge (vs no papillae as in Oxydromus, Hesione, etc.; ~20 papillae as in Micropodarke) and bearing four pairs of tentacular cirri on both sides of the peristomium (vs three pairs as in Oxydromus, Micropodarke, Syllidia, etc.) (Pleijel 1998;Rizzo and Salazar-Vallejo 2014). Therefore, it is not difficult to identify specimens to the genus level.
The genus Podarkeopsis currently includes only nine valid species, and in this study we describe a tenth species, P. chinensis sp. nov., the first Podarkeopsis species described from the Indo-Pacific. Podarkeopsis cincinnata (Verrill, 1881) collected from New England, USA, has three pairs (instead of four as in other species of Podarkeopsis) of slender tentacular cirri on each side and about 12 papillae according to the original description; therefore, it is considered here an invalid species of Podarkeopsis, and its status could not be determined without checking the type material. In addition to P. chinensis sp. nov., we also found some other species of Podarkeopsis from the China seas, which indicated an underestimated species diversity of this genus. Given that many undescribed species of Podarkeopsis and several species in this region are potentially misidentified, we predict that further studies may lead to the discovery of more species.
In addition to the mentioned characteristics, we also noted two, minute teeth on the inner wall of the pharynx in all specimens of P. chinensis sp. nov. examined ( Fig. 2H-L), but these teeth have not been reported from the other species of Podarkeopsis. Besides, we observed a special type of hooded neurochaetae that had not been reported from other species of Podarkeopsis, but a similar kind of chaeta had been noted from some other hesionid species (Perkins 1984: 579;Wang et al. 2018: fig. 3O, P; this study, Fig. 4F2, H2), Thus, further studies should explore the use of these additional characters to distinguish species in the genus.